Pon1 as a marker for heart failure

ABSTRACT

Provided herein are methods for assessing the risk a test subject with heart failure has of experiencing a major adverse cardiac event, requiring revascularization, requiring a heart transplant, requiring unscheduled hospitalization for heart failure, progression of heart failure status, or any combination thereof. Also provided herein are methods for assessing the risk a test subject has of developing heart failure. The present methods comprise determining the levels of paraoxonase 1 activity in the serum, non-chelated plasma, or both in the test subject and comparing the level of PON1 activity in the test subject&#39;s sample with a control or baseline value based on levels of PON1 activity in serum, non-chelated plasma, or both samples from a population of control subjects. Also provided herein are kits useful in assessing such risks.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/235,242, filed Aug. 19, 2009, and U.S. Provisional Application No.61/286,681, filed Dec. 15, 2009, both of which are incorporated hereinby reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported by U.S. National Institutes of Health grants P01HL076491-055328, P01 HL087018-020001, and P01 HL077107-050004. The U.S.government has certain rights in this invention.

BACKGROUND

The present disclosure relates to methods which can be used to determinewhether a test subject with heart failure, more particularly a humansubject, is at a low risk or high risk of experiencing a major adversecardiovascular event. It also relates to methods which can be used todetermine whether a test subject without heart failure is at a low orhigh risk of developing heart failure. Also disclosed herein are kitsuseful for determining suck risks.

Heart failure (HF) is generally defined as inability of the heart tosupply sufficient blood flow to meet the body's needs. Common causes ofheart failure include myocardial infarction (heart attacks) and otherforms of ischemic heart disease, hypertension, valvular heart disease,and cardiomyopathy. Heart failure can cause a number of symptomsincluding shortness of breath, coughing, chronic venous congestion,ankle swelling, and exercise intolerance. Heart failure is oftenundiagnosed due to a lack of a universally agreed definition andchallenges in definitive diagnosis. Treatment commonly consists oflifestyle measures (such as decreased salt intake) and medications, andsometimes devices or even surgery.

Heart failure is a common, costly, disabling, and potentially deadlycondition. In developing countries, around 2% of adults suffer fromheart failure, but in those over the age of 65, this increases to 6-10%.Mostly due to costs of hospitalization it is associated with a highhealth expenditure; costs have been estimated to amount to 2% of thetotal budget of the National Health Service in the United Kingdom, andmore than $35 billion in the United States. Heart failure is associatedwith significantly reduced physical and mental health, resulting in amarkedly decreased quality of life. With the exception of heart failurecaused by reversible conditions, the condition usually worsens withtime. Although some patients survive many years, progressive disease isassociated with an overall annual mortality rate of 10%. Thus, there isa need for methods of determining the risk for experiencing a majoradverse cardiac event in a broader spectrum of individuals.

SUMMARY

The present disclosure provides new methods for assessing the risk ofexperiencing a major adverse cardiac event (MACE) in a test subject withheart failure. The methods comprise determining the level of paraoxonase1 (PON1) activity in a serum or non-chelated plasma sample from the testsubject with heart failure and comparing the level of PON1 activity inthe test subject's serum or non-chelated plasma sample with a control orbaseline value. The control or baseline value is based on levels of PON1activity in serum or non-chelated plasma samples from a population ofcontrol subjects. A test subject whose level of PON1 activity is lessthan the control or baseline value is at risk of experiencing a MACE.

The present disclosure provides new methods for assessing the risk ofdeveloping heart failure in a test subject. In some embodiments the testsubject is apparently healthy and does not have heart failure. Themethods comprise determining the level of paraoxonase 1 (PON1) activityin a serum or non-chelated plasma sample from the test subject andcomparing the level of PON1 activity in the test subject's serum ornon-chelated plasma sample with a control or baseline value. The controlor baseline value is based on levels of PON1 activity in serum ornon-chelated plasma samples from a population of control subjects. Atest subject whose level of PON1 activity is less than the control orbaseline value is at risk of developing heart failure.

As used herein the term “major adverse cardiac event” includes, but isnot limited to, non-fatal cerebrovascular accident, non-fatal myocardialinfarction, death, or a combination thereof. In some embodiments, heartfailure includes, but is not limited to, systolic heart failure,diastolic heart failure, left ventricular dysfunction, or combinationsthereof. In some embodiments, the left ventricular dysfunction ischaracterized by having a lower than normal left ventricular ejectionfraction (LVEF). In some embodiments, the LVEF is less than 40%. In someembodiments, non-fatal cerebrovascular accident includes, but is notlimited to, stroke, transient ischemic attacks, or both.

In some embodiments, PON1 activity is determined by measuringarylesterase, paraoxonase, lipolactonase, lactonase, or combinations ofthese activities. These activities can be determined using knownmethods, including, but not limited to, spectrophotometry. In someembodiments, the spectrophotometry can be chosen from the groupconsisting of visible, ultraviolet, and fluorescence basedspectrophotometry.

In some embodiments of the methods, the test subject has no evidence ofcardiovascular disease (CVD) or heart failure. As used herein, “evidenceof cardiovascular disease” means that the test subject, individual, orpatient has no history of CVD and/or less than 50% stenosis of all majorcoronary vessels. In some embodiments, the control subjects areapparently healthy control subjects. In some embodiments, the apparentlyhealthy control subjects are without any known CVD.

Also disclosed herein are methods of determining if the test subject isat near term risk of experiencing a major adverse cardiac event. As usedherein, the term “near term” means within three years. Thus, subjectswho are at near term risk may be at risk of experiencing a major adversecardiac event within the following day, 3 months, 6 months, or one yearafter being tested. In some embodiments the methods are useful fordetermining if the test subject is at long term risk of experiencing amajor adverse cardiac event. As used herein, the term “long term” meanswithin ten years. Thus, subjects who are at long term risk may be atrisk of experiencing a major adverse cardiac event within the followingfive years, seven years, 10 years, or longer after being tested. Thepresent methods are especially useful for identifying those individualswho are at high risk and thus, in need of highly aggressive therapies aswell as those individuals who require no therapies targeted atpreventing such events.

Also disclosed herein are methods for assessing the risk of the testsubject with heart failure requiring revascularization. The methodscomprise determining the level of paraoxonase 1 (PON1) activity in aserum or non-chelated plasma sample from the test subject and comparingthe level of PON1 activity in the test subject's serum or non-chelatedplasma sample with a control or baseline value. The control or baselinevalue is based on levels of PON1 activity in serum or non-chelatedplasma samples from a population of control subjects. A test subjectwhose level of PON1 activity is less than the control or baseline valueis at risk of requiring revascularization.

In some embodiments, PON1 activity is determined by measuringarylesterase, paraoxonase, lipolactonase, lactonase, or combinations ofthese activities. These activities can be determined using knownmethods, including, but not limited to, spectrophotometry. In someembodiments, the spectrophotometry can be chosen from the groupconsisting of visible, ultraviolet, and fluorescence basedspectrophotometry.

In some embodiments of the methods, the test subject has no evidence ofcardiovascular disease (CVD) or heart failure. As used herein, “evidenceof cardiovascular disease” means that the test subject, individual, orpatient has no history of CVD and/or less than 50% stenosis of all majorcoronary vessels. In some embodiments, the control subjects areapparently healthy control subjects. In some embodiments, the apparentlyhealthy control subjects are without any known CVD.

Also disclosed herein are methods of determining if the test subject isat near term risk of requiring revascularization. As used herein, theterm “near term” means within three years. Thus, subjects who are atnear term risk may be at risk of requiring revascularization within thefollowing day, 3 months, 6 months, or one year after being tested. Insome embodiments the methods are useful for determining if the testsubject is at long term risk of requiring revascularization. As usedherein, the term “long term” means within ten years. Thus, subjects whoare at long term risk may be at risk of requiring revascularizationwithin the following five years, seven years, 10 years, or longer afterbeing tested. The present methods are especially useful for identifyingthose individuals who are at high risk and thus, in need of highlyaggressive therapies as well as those individuals who require notherapies targeted at preventing such events.

Also disclosed herein are methods for assessing the risk of the testsubject with heart failure requiring a heart transplant. The methodscomprise determining the level of paraoxonase 1 (PON1) activity in aserum or non-chelated plasma sample from the test subject and comparingthe level of PON1 activity in the test subject's serum or non-chelatedplasma sample with a control or baseline value. The control or baselinevalue is based on levels of PON1 activity in serum or non-chelatedplasma samples from a population of control subjects. A test subjectwhose level of PON1 activity is less than the control or baseline valueis at risk of requiring a heart transplant.

In some embodiments, PON1 activity is determined by measuringarylesterase, paraoxonase, lipolactonase, lactonase, or combinations ofthese activities. These activities can be determined using knownmethods, including, but not limited to, spectrophotometry. In someembodiments, the spectrophotometry can be chosen from the groupconsisting of visible, ultraviolet, and fluorescence basedspectrophotometry.

In some embodiments of the methods, the test subject has no evidence ofcardiovascular disease (CVD) or heart failure. As used herein, “evidenceof cardiovascular disease” means that the test subject, individual, orpatient has no history of CVD and/or less than 50% stenosis of all majorcoronary vessels. In some embodiments, the control subjects areapparently healthy control subjects. In some embodiments, the apparentlyhealthy control subjects are without any known CVD.

Also disclosed herein are methods of determining if the test subject isat near term risk of requiring a heart transplant. As used herein, theterm “near term” means within three years. Thus, subjects who are atnear term risk may be at risk of requiring a heart transplant within thefollowing day, 3 months, 6 months, or one year after being tested. Insome embodiments the methods are useful for determining if the testsubject is at long term risk of requiring a heart transplant. As usedherein, the term “long term” means within ten years. Thus, subjects whoare at long term risk may be at risk of requiring a heart transplantwithin the following five years, seven years, 10 years, or longer afterbeing tested. The present methods are especially useful for identifyingthose individuals who are at high risk and thus, in need of highlyaggressive therapies as well as those individuals who require notherapies targeted at preventing such events.

Also disclosed herein are methods for assessing the risk of the testsubject with heart failure requiring unscheduled hospitalization forheart failure. The methods comprise determining the level of paraoxonase1 (PON1) activity in a serum or non-chelated plasma sample from the testsubject and comparing the level of PON1 activity in the test subject'sserum or non-chelated plasma sample with a control or baseline value.The control or baseline value is based on levels of PON1 activity inserum or non-chelated plasma samples from a population of controlsubjects. A test subject whose level of PON1 activity is less than thecontrol or baseline value is at risk of requiring unscheduledhospitalization for heart failure.

In some embodiments, PON1 activity is determined by measuringarylesterase, paraoxonase, lipolactonase, lactonase, or combinations ofthese activities. These activities can be determined using knownmethods, including, but not limited to, spectrophotometry. In someembodiments, the spectrophotometry can be chosen from the groupconsisting of visible, ultraviolet, and fluorescence basedspectrophotometry.

In some embodiments of the methods, the test subject has no evidence ofcardiovascular disease (CVD) or heart failure. As used herein, “evidenceof cardiovascular disease” means that the test subject, individual, orpatient has no history of CVD and/or less than 50% stenosis of all majorcoronary vessels. In some embodiments, the control subjects areapparently healthy control subjects. In some embodiments, the apparentlyhealthy control subjects are without any known CVD.

Also disclosed herein are methods of determining if the test subject isat near term risk of requiring unscheduled hospitalization for heartfailure. As used herein, the term “near term” means within three years.Thus, subjects who are at near term risk may be at risk of requiringunscheduled hospitalization for heart failure within the following day,3 months, 6 months, or one year after being tested. In some embodimentsthe methods are useful for determining if the test subject is at longterm risk of requiring unscheduled hospitalization for heart failure. Asused herein, the term “long term” means within ten years. Thus, subjectswho are at long term risk may be at risk of requiring unscheduledhospitalization for heart failure within the following five years, sevenyears, 10 years, or longer after being tested. The present methods areespecially useful for identifying those individuals who are at high riskand thus, in need of highly aggressive therapies as well as thoseindividuals who require no therapies targeted at preventing such events.

Also disclosed herein are methods for assessing the risk of progressionof heart failure in the test subject. The methods comprise determiningthe level of paraoxonase 1 (PON1) activity in a serum or non-chelatedplasma sample from the test subject and comparing the level of PON1activity in the test subject's serum or non-chelated plasma sample witha control or baseline value. The control or baseline value is based onlevels of PON1 activity in serum or non-chelated plasma samples from apopulation of control subjects. A test subject whose level of PON1activity is less than the control or baseline value is at risk ofprogression of heart failure.

In some embodiments, PON1 activity is determined by measuringarylesterase, paraoxonase, lipolactonase, lactonase, or combinations ofthese activities. These activities can be determined using knownmethods, including, but not limited to, spectrophotometry. In someembodiments, the spectrophotometry can be chosen from the groupconsisting of visible, ultraviolet, and fluorescence basedspectrophotometry.

In some embodiments of the methods, the test subject has no evidence ofcardiovascular disease (CVD) or heart failure. As used herein, “evidenceof cardiovascular disease” means that the test subject, individual, orpatient has no history of CVD and/or less than 50% stenosis of all majorcoronary vessels. In some embodiments, the control subjects areapparently healthy control subjects. In some embodiments, the apparentlyhealthy control subjects are without any known CVD.

Also disclosed herein are methods of determining if the test subject isat near term risk of progression of heart failure. As used herein, theterm “near term” means within three years. Thus, subjects who are atnear term risk may be at risk of progression of heart failure within thefollowing day, 3 months, 6 months, or one year after being tested. Insome embodiments the methods are useful for determining if the testsubject is at long term risk of progression of heart failure. As usedherein, the term “long term” means within ten years. Thus, subjects whoare at long term risk may be at risk of progression of heart failurewithin the following five years, seven years, 10 years, or longer afterbeing tested. The present methods are especially useful for identifyingthose individuals who are at high risk and thus, in need of highlyaggressive therapies as well as those individuals who require notherapies targeted at preventing such events.

Also disclosed herein are kits useful in assessing whether a subject isat risk of at least one of the following: experiencing a MACE; requiringrevascularization; requiring a heart transplant; requiring unscheduledhospitalization for heart failure; and progression of heart failurestatus. The kit may comprise one or more reagents for determining thelevel of PON1 activity in a subject. In some embodiments, PON1 activityis determined by measuring arylesterase, paraoxonase, lipolactonase,lactonase, or combinations of these activities. These activities can bedetermined using known methods, including, but not limited to,spectrophotometry. In some embodiments, the spectrophotometry can bechosen from the group consisting of visible, ultraviolet, andfluorescence based spectrophotometry. In some embodiments the kit mayfurther comprise information for assessing the subject's risk of atleast one of the following: experiencing a MACE; requiringrevascularization; requiring a heart transplant; requiring unscheduledhospitalization for heart failure; and progression of heart failurestatus.

In some embodiments of the methods, the test subject has no evidence ofcardiovascular disease (CVD) or heart failure. As used herein, “evidenceof cardiovascular disease” means that the test subject, individual, orpatient has no history of CVD and/or less than 50% stenosis of all majorcoronary vessels. In some embodiments, the control subjects areapparently healthy control subjects. In some embodiments, the apparentlyhealthy control subjects are without any known CVD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of serum arylesterase activity between healthycontrols (n=300), patients with stable systolic heart failure (n=760),and hospitalized patients with advanced decompensated heart failure(n=75).

FIG. 2 shows Kaplan-Meier analysis for long-term major adverse cardiacevents stratified by serum arylesterase quartiles (Log-rank, p=0.027);quartile ranges as followed: Q1: <82 μM/min/mL; Q2: 82-97 μM/min/mL; Q3:98-116; Q4: >116.

FIG. 3 shows Kaplan-Meier analysis for long-term major adverse cardiacevents stratified by optimal cut-off of 121 μM/min/mL (Log-rank,p=0.006).

DETAILED DESCRIPTION

The present disclosures will now be described by reference to moredetailed embodiments, with occasional reference to the accompanyingdrawings. These disclosures may, however, be embodied in different formsand should not be construed as limited to the embodiments set forthherein. Rather these embodiments are provided so that this disclosurewill be thorough and complete, and will convey the scope of thedisclosures to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which these disclosures belong. The terminology used in thedescription herein is for describing particular embodiments only and isnot intended to be limiting of the disclosures. As used in thedescription and the appended claims, the singular forms “a,” “an,” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,concentrations, and so forth as used in the specification and claims areto be understood as being modified in all instances by the term “about,”unless the context indicates otherwise. Accordingly, unless otherwiseindicated, the numerical properties set forth in the followingspecification and claims are approximations that may vary depending onthe desired properties sought to be obtained in embodiments of thepresent disclosures. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of the disclosures areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical values, however,inherently contain certain errors necessarily resulting from error foundin their respective measurements.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Provided herein are methods of assessing the risk of experiencing amajor adverse cardiac event in a subject with heart failure. The methodscomprise determining the levels of PON1 activity in a serum,non-chelated plasma, or both types of sample from the subject and arebased on the discovery that subjects who have decreased systemic levelsof PON1 activity relative to a control or representative value are atgreater risk of experiencing a major adverse cardiac that subjects whosesystemic levels of PON1 activity are higher relative to a control orrepresentative value.

PON1 enzymatic activity in the subject's serum, non-chelated plasma, orboth types of sample can be determined using a number of differentsubstrates. Thus, in one embodiment PON1 enzymatic activity can bedetermined by measuring arylesterase activity (as described below) inthe sample obtained from the test subject. In another embodiment PON1enzymatic activity can be determined by measuring paraoxonase activity(as described below) in the sample obtained from the test subject. Inanother embodiment PON1 enzymatic activity can be determined bymeasuring lipolactonase activity in the sample obtained from the testsubject. Methods for measuring such activity are described in GaidukovL, Tawfik DS. “The development of human sera tests for HDL-bound serumPON1 and its lipolactonase activity” J Lipid Res. 2007 Jul;48(7):1637-46. In another embodiment PON1 enzymatic activity can bedetermined by measuring lactonase activity in the sample obtained fromthe test subject. Methods of measuring lactonase activity are known inthe art. The level of PON1 activity in the sample can be determined bymeasuring the PON1 enzymatic activity in the sample and normalizing thisvalue to obtain the PON1 activity or mass per milliliter of serum, permilliliter of plasma, or per weight.

Preparation of Bodily Sample.

Suitable sources for the bodily samples include but are not limited towhole blood samples, samples of blood fractions, including but notlimited to serum and non-chelated plasma. The sample source may be freshblood or stored blood (e.g., in a blood bank) or blood fractions. Thesample source may be a blood sample expressly obtained for the assaysdisclosed herein or a blood sample obtained for another purpose whichcan be sub-sampled for the assays disclosed herein.

In one embodiment, the biological sample source is whole blood. Wholeblood may be obtained from the subject using standard clinicalprocedures. In another embodiment, the biological sample source isplasma. Plasma may be obtained from whole blood samples bycentrifugation of anti-coagulated blood containing anti-coagulants suchas heparin. However, it is preferred that such samples do not contain ametal ion chelator such as EDTA since these types of compounds caninhibit PON1 enzymatic activity. Such process provides a buffy coat ofwhite cell components and a supernatant of the plasma. In anotherembodiment, the biological sample is serum. Serum may be obtained bycentrifugation of whole blood samples that have been collected in tubesthat are free of anti-coagulant. The blood is permitted to clot prior tocentrifugation. The yellowish-reddish fluid that is obtained bycentrifugation is the serum.

The sample may be pretreated as necessary by dilution in an appropriatebuffer solution, heparinized, concentrated if desired, or fractionatedby any number of methods including but not limited toultracentrifugation, fractionation by fast performance liquidchromatography (FPLC), or precipitation of apolipoprotein B containingproteins with dextran sulfate or other methods. Any of a number ofstandard aqueous buffer solutions, employing one of a variety ofbuffers, such as phosphate, Tris, or the like, at physiological pH canbe used.

Control/Representative Value.

The level of PON1 activity in the bodily sample obtained from the testsubject is compared to a control value. The control value is based uponthe levels of PON1 activity in comparable samples obtained from thegeneral population or from a select population of human subjects. Forexample the control value may be derived from serum and/or non-chelatedplasma samples obtained from apparently healthy individuals who have notpreviously had any signs or symptoms indicating the presence ofatherosclerosis, such as angina pectoris, history of an acute adversecardiovascular event such as a myocardial infarction or stroke, evidenceof atherosclerosis by diagnostic imaging methods including, but notlimited to coronary angiography. In certain embodiments, the controlvalue may be derived from subjects who have not experienced a majoradverse cardiac event within a predetermined period of time, e.g. one tothree years, after providing the control sample. In another example, thecontrol value can be derived from an apparently healthy nonsmokerpopulation. “Nonsmoker,” as used herein, means an individual who, at thetime of the evaluation, is not a smoker. This includes individuals whohave never smoked as well as individuals who in the past have smoked buthave not smoked in the previous 12 months. An apparently healthy,nonsmoker population may have a different normal range of PON1 enzymaticactivity than will a smoking population. Accordingly, the control valuesselected may take into account the category into which the test subjectfalls. Appropriate categories can be selected with no more than routineexperimentation by those of ordinary skill in the art.

In certain embodiments, the control value can be a baseline valuederived by measuring systemic PON1 levels in the subject himself at anearlier time. Thus, the present method can be used to monitor over timethe subject's risk of experiencing a major adverse cardiac event.

The control value can take a variety of forms. The control value can bea single cut-off value, such as a median or mean. The control value canbe established based upon comparative groups such as where the risk inone defined group is double the risk in another defined group. Thecontrol can be a range, for example, where the general population isdivided equally (or unequally) into groups, such as a low risk group, amedium risk group and a high-risk group, or into tertiles or quadrants,the lowest tertile or quadrant being individuals with substantiallyaltered risk compared to individuals in the highest tertile or quadrant.In the case of paraoxonase enzymatic activity, which is inverselycorrelated with cardiac risks, subjects in the lowest tertile orquartile of paraoxonase enzymatic activity have the highest risk, andsubjects in the highest tertile or quartile of paraoxonase enzymaticactivity have the lowest risk.

Control values of PON1 enzymatic activity, such as for example, meanlevels, median levels, or “cut-off” levels, are established by assayinga large sample of individuals in the general population or the selectpopulation and using a statistical model such as the predictive valuemethod for selecting a positivity criterion or receiver operatorcharacteristic curve that defines optimum specificity (highest truenegative rate) and sensitivity (highest true positive rate) as describedin Knapp, R. G., and Miller, M. C. (1992). Clinical Epidemiology andBiostatistics. William and Wilkins, Harual Publishing Co. Malvern, Pa.,which is specifically incorporated herein by reference. A “cutoff” valuecan be determined for each risk predictor that is assayed.

The levels of PON1 enzymatic activity in the individual's bodily samplemay be compared to a single control value or to a range of controlvalues. If the level of PON1 enzymatic activity in the test subject'sbodily sample is lower than the control value or range of controlvalues, the test subject is at greater risk of experiencing a majorcardiac event than individuals with levels comparable to or above thecontrol value or control range of values. The extent of the differencebetween the test subject's risk predictor levels and control value isalso useful for characterizing the extent of the risk and thereby,determining which individuals would most greatly benefit from certainaggressive therapies. In those cases, wherein the control value rangesare divided into a plurality of groups, such as the control value rangesfor individuals at high risk, average risk, and low risk, the comparisoninvolves determining into which group the test subject's level of therelevant risk predictor falls.

The present methods are useful for determining if and when certaintherapies, including therapeutic agents, which are targeted atpreventing major adverse cardiac events should and should not beprescribed for a patient. For example, individuals with values of PON1activity below a certain cutoff value, or that are in the lower tertileor quartile of a “normal range,” could be identified as those in need ofmore aggressive intervention.

The present methods are also useful for identifying individuals atincreased risk of experiencing a major cardiovascular event who mightotherwise not have been identified by existing screeningprotocols/methods.

In another aspect, the present disclosure provides methods ofdetermining the risk for requiring revascularization as defined byangioplasty, stenting, and coronary artery bypass grafting (CABG) in asubject. In some embodiments the subject has no evidence of coronaryartery disease. The method comprises determining the levels of PON1enzymatic activity in the subject's serum, non-chelated plasma, or both.A subject whose levels of PON1 activity are low relative to a controlvalue based on levels of PON1 activity in samples from the generalpopulation or a select population is a greater risk of requiringrevascularization than a subject whose systemic levels of PON 1enzymatic activity are high relative to the control value.

Also disclosed herein are methods of determining the risk for requiringa heart transplant in a subject. In some embodiments the subject has noevidence of coronary artery disease. The method comprises determiningthe levels of PON1 enzymatic activity in the subject's serum,non-chelated plasma, or both. A subject whose levels of PON1 activityare low relative to a control value based on levels of PON 1 activity insamples from the general population or a select population is a greaterrisk of requiring a heart transplant than a subject whose systemiclevels of PON1 enzymatic activity are high relative to the controlvalue.

Also disclosed herein are methods of determining the risk unscheduledhospitalization for heart failure in a subject. In some embodiments thesubject has no evidence of coronary artery disease. The method comprisesdetermining the levels of PON1 enzymatic activity in the subject'sserum, non-chelated plasma, or both. A subject whose levels of PON1activity are low relative to a control value based on levels of PON1activity in samples from the general population or a select populationis a greater risk of requiring unscheduled hospitalization for heartfailure than a subject whose systemic levels of PON 1 enzymatic activityare high relative to the control value.

Also disclosed herein are methods of determining the risk progression ofheart failure status in a subject. In some embodiments the subject hasno evidence of coronary artery disease. The method comprises determiningthe levels of PON1 enzymatic activity in the subject's serum,non-chelated plasma, or both. A subject whose levels of PON1 activityare low relative to a control value based on levels of PON1 activity insamples from the general population or a select population is a greaterrisk of progression of heart failure status than a subject whosesystemic levels of PON1 enzymatic activity are high relative to thecontrol value.

In another aspect, the present disclosure provides methods ofdetermining whether a subject who has experienced a major adversecardiac event is at risk of experiencing a recurrent majorcardiovascular event, e.g., reinfarction. The method comprisesdetermining the levels of PON1 activity in the subject's sample. Asubject whose levels of PON1 activity are low relative to a controlvalue based on levels of PON1 activity in samples from the generalpopulation or a select population is a greater risk of experiencingrecurrent or subsequent major adverse cardiac event than a subject whosesystemic levels of PON1 enzymatic activity are high relative to thecontrol value.

Examples

The following examples are for illustration only and are not intended tolimit the scope of the disclosures.

Diminished serum arylesterase activity, catalyzed by the HDL-associatedparaoxonase-1 (PON1), is associated with heightened systemic oxidativestress and atherosclerosis risk. The role of serum arylesterase activityin systolic heart failure, particularly in relation with establishedcardiac biomarkers, is unknown.

Serum arylesterase activity was measured in 760 subjects with impairedleft ventricular systolic function (left ventricular ejection fraction[LVEF] <50%), and the subjects were prospectively followed for majoradverse cardiac events (MACE, including for example death, non-fatalmyocardial infarction, and stroke) for 3 years. In the study cohort(mean age 64±11 years, 74% male, median LVEF 35%, median creatinineclearance [CrCl] 87 mg/dL), mean serum arylesterase activity was lowerthan healthy controls but higher than those with advanced decompensatedheart failure (mean 98 ±25 μmol/min/mL). Within the cohort, there wasmodest inverse correlation between serum arylesterase activity andB-type natriuretic peptide (BNP, r=-0.23, p<0.01), and weak correlationswere observed between serum arylesterase activity and LVEF (Spearman'sr=0.12, p<0.01) and high-sensitivity C-reactive protein (hsCRP, r=-0.13,p<0.01). Lower serum arylesterase activity was a strong predictor ofpoorer outcomes (Quartile 4 versus Quartile 1, Hazard ratio 3.27, 95%confidence intervals 1.66-6.44, p<0.001). After adjusting for log(BNP),log(hsCRP), Framingham risk factors, and CrCl, the lowest threequartiles of arylesterase still maintain a 2.6-fold increased risk inMACE at 3 years (Q4 vs. lower 3 quartiles, Hazard ratio 2.59 [1.34,4.99], p=0.004).

In patients with systolic heart failure, decreased PON1 activity, an HDLassociated protein with a strong link to systemic indices of oxidantstress, predicts higher long-term adverse cardiac event ratesindependent of established clinical and biochemical risk factors.

Introduction

Oxidative stress plays an important role in the pathogenesis andprogression of heart failure^(1,2). Different degrees of oxidativestress are operative in the syndrome of heart failure and cardiacdysfunction, ranging from direct damage to cellular proteins andmembranes (leading to apoptosis and necrosis), to alterations in cardiacenergetics or extracellular matrix remodeling. Measures of stableoxidative byproducts, including oxidized low-density lipoproteins(LDL)⁷, malondialdehyde⁸, isoprostaines⁹, and urinary bioptyrins¹⁰, areincreased in the setting of heart failure. However, reactive oxidantspecies and their byproducts are often transient, compartmentalized, anddifficult to quantify in vivo, which has led to the search for indirectmarkers of oxidative stress.

Increased oxidative stress results from an imbalance between reactiveoxygen, nitrogen, and halogenating species and endogenous antioxidantdefense mechanisms to scavenge free radicals and their byproducts^(4,5),such as superoxide dismutase, catalase, glutathione peroxidase³.Therefore, an imbalance between oxidative and anti-oxidative mechanismsmay lead to deleterious consequences⁶. Paraoxonase-1 (PON1) is a highdensity lipoprotein (HDL)-associated glycoprotein believed to play a keyrole facilitating systemic anti-oxidant activities of HDL, including theremodeling of oxidized phospholipids^(11,12). PON1 is produced by theliver, and catalyzes calcium-dependent esterase and lactonase activitiescritical to its lipid remodeling and systemic anti-oxidant functions.Numerous studies have shown that PON1 serves as a primary contributor tosystemic (serum) arylesterase activity (hydrolase activity on carboxylicester bonds such as phenyl acetate)¹³. Systemic measures of PON1activity, such as serum arylesterase activity, have been shown to havestrong correlations with multiple systemic measures of oxidant stress,including multiple distinct fatty acid oxidation products quantified byliquid chromatography with on-line stable isotope dilution tandem massspectrometryl³. Thus, both human clinical investigations¹³, and studiesemploying PON1 knockout mice (Shih DM, Gu L, Xia YR, Navab M, Li W F,Hama S, Castellani L W, Furlong C E, Costa L G, Fogelman A M, Lusis A J.Nature. 1998 Jul 16;394(6690):284-7. Rozenberg O, Rosenblat M, ColemanR, Shih D M, Aviram M. Free Radic Biol Med. 2003 Mar 15;34(6):774-84.)are consistent with PON1 serving a major anti-oxidant function in vivo.Herein, the potential role of serum PON1 activity was examined, asmonitored by arylesterase activity measurements, as a predictor ofadverse disease progression among patients with systolic heart failure.

Methods

Study population. The Cleveland Clinic GeneBank study is a large(n>10,000), prospective cohort study from 2001-2006 that established awell-characterized clinical repository with clinical and longitudinaloutcomes data comprised from consenting subjects undergoing electivediagnostic cardiac catheterization procedure not in the setting of acutecoronary syndrome. All GeneBank participants gave written informedconsent approved by the Cleveland Clinic Institutional Review Board.Clinical outcomes were prospectively ascertained over the ensuing 3years for all subjects following enrollment. Major adversecardiovascular event (MACE) was defined as all-cause mortality,non-fatal myocardial infarction, or non-fatal cerebrovascular accidentfollowing enrollment.

This analysis included 760 consecutive subjects with stable systolicheart failure (left ventricular ejection fraction [LVEF] <50% asdetermined by echocardiography, radionuclide or contrastventriculography) enrolled in GeneBank with serum samples available foranalysis. The Framingham Risk Score was calculated for each subject asstandard cardiovascular risk factors. An estimate of creatinineclearance (CrCl) was calculated using the Cockcroft-Gault equation. Highsensitivity C-reactive protein (hsCRP), B-type natriuretic peptide(BNP), creatinine, and fasting blood glucose and lipid profiles weremeasured on the Abbott Architect platform (Abbott Laboratories, AbbottPark Ill.).

Serum arylesterase activity assay. Serum arylesterase activity wasmeasured by UV spectrophotometry in a 96-well plate format (Spectramax384 Plus, Molecular Devices, Sunnyvale, Calif.) using phenyl acetate(Sigma-Aldrich, St Louis, Miss.) as substrate. Briefly, initialhydrolysis rates were determined at 270 nm in 50-fold diluted serum(final) in reactions mixtures composed of 3.4 mM phenylacetate, 9 mMTris hydrochloride, pH 8, and 0.9 mM calcium chloride at 24° C. Anextinction coefficient (at 270 nm) of 1310 M-1·cm-1 was used forcalculating units of arylesterase activity, which are expressed as theamount of phenyl acetate hydrolyzed in μM/min/mL of serum. Theintra-assay and inter-assay coefficients of variance for performance ofarylesterase were 1.2% and 3.9%, respectively, on 20 replicatesperformed on 10 different days.

To examine the range of serum arylesterase activity using this assay, across-sectional comparison was performed between the cohort of stablesystolic heart failure with a separate set of apparently healthy controlsubjects without known cardiac diseases from a health screen (n=300), aswell as a group of consecutive patients with advanced decompensatedheart failure admitted to the heart failure intensive care unit forhemodynamically-guided therapy including intravenous diuretic therapy(n=73), all with written informed consent approved by the ClevelandClinic Institutional Review Board.

Statistical analyses. The Student's t-test or Wilcoxon-Rank sum test forcontinuous variables and chi-square test for categorical variables wereused to examine the difference between the groups. Receiver OperatorCharacteristic (ROC) curve analyses were performed to determine theoptimal cutoff at 121 μM/min/mL, with risk of event estimated using5-fold cross-validation by a Cox model and logistic regression model.Adjustments were made for individual traditional cardiac risk factor,Framingham Risk Score, log-transformed hsCRP, log-transformed BNP, andCrCl to predict incident 3-year MACE risks. Kaplan-Meier analysis withCox proportional hazards regression was used for time-to-event analysisto determine Hazard ratio (HR) and 95% confidence intervals (95% CI) forMACE. All analyses were performed using SAS version 8.2 (Cary, N.C.) andR 2.8.0 (Vienna, Austria). P values <0.05 were considered statisticallysignificant.

Results

Study population. Table 1 describes the baseline characteristics of thesubjects, which is a relatively well-compensated patient cohort withmedian systolic ejection fraction of 35%, normal mean creatinineclearance of 100±41 ml/min/1.73 m², 63% ACE inhibitor use, and 40%treated with standing diuretic therapy. Serum arylesterase levels werenormally distributed, with a mean of 98±25 μM/min/mL, which was lowerthan that in apparently healthy control population (mean 115±26μM/min/mL, p<0.01, FIG. 1), but higher than that of hospitalizedpatients with advanced systolic heart failure (mean 69±22 μM/min/mL,p<0.01). In general, male subjects tended to have lower serumarylesterase activity levels than female subjects.

TABLE 1 Baseline Subject Characteristics. Variable Value Age (years) 64± 11 Male (%) 74 Diabetes mellitus (%) 40 Hypertension (%) 72 Ischemicetiology (%) 68 LDL cholesterol (mg/dL) 101 (81, 120) HDL cholesterol(mg/dL) 32 (26, 38) Triglycerides (mg/dL) 119 (87, 167) B-typenatriuretic peptide (mg/dL) 193 (86, 481) hsCRP (mg/L)  2.7 (1.2, 6.9)LV Ejection Fraction (%-units) 35 (30, 45) Creatinine clearance(ml/min/1.73 m²) 100 ± 41  Aspirin (%) 73 Statins (%) 62 Diuretics (%)40 ACE inhibitors (%) 63 Beta-blockers (%) 67

Values expressed in mean±standard deviation or median (interquartilerange). Abbreviations: cTnI=LDL=low-density lipoprotein;HDL=high-density lipoprotein; hsCRP=high-sensitivity C-reactive protein;ATP III=Adult Treatment Panel III guidelines.

Serum arylesterase levels and cardiac biomarkers. There was modestinverse correlation between serum arylesterase and plasma B-typenatriuretic peptide levels (BNP, r=-0.23, p<0.01). In contrast, weakercorrelations were observed between arylesterase levels and LVEF(Spearman's r=0.12, p<0.01) and high-sensitivity C-reactive protein(hsCRP, r=-0.13, p<0.01). In logistic regression models, male gender,BNP, and hsCRP were independent predictors of elevated serumarylesterase levels (Table 2).

TABLE 2 Logistic regression model for determinants of serum arylesteraselevels. Standard Estimate Error z-value p-value Age −0.001 0.009 −0.1280.898 Gender −0.634 0.189 −3.358 <0.001 Creatinine clearance 0.002 0.0030.950 0.342 BNP −0.332 0.076 −4.380 <0.001 LV ejection fraction 0.0050.009 0.559 0.576 Diabetes mellitus −0.104 0.160 −0.649 0.517Hypertension 0.209 0.174 1.206 0.228 hsCRP −0.009 0.004 −2046 0.04Abbreviations: BNP = B-type natriuretic peptide; LV = left ventricle;hsCRP = high-sensitivity C-reactive protein.

Serum arylesterase levels and major adverse cardiac outcomes. A total of134 events (non-fatal MI, stroke or death) were recorded within the3-year period of follow-up. Lower serum arylesterase activity wasassociated with poorer long-term outcomes when stratified by quartiles(Hazard ratios [95% confidence interval] for Q4 vs. Q3: 1.99[1.16-3.41], p=0.01; Q4 vs. Q2: 1.87 [1.08-3.24], p=0.02; Q4 vs. Q1:2.18 [1.28, 3.73], p<0.001). When Kaplan-Meier analysis was performed(stratified according to quartiles) there was clear distinction betweenthe highest quartile (cut-off at 116 μM/min/mL) and the lower threequartiles (HR 1.98 [1.23, 3.18], p=0.005, FIG. 2). Kaplan-Meier analysisdemonstrated heightened risk associated with the development of MACE at3 years when stratified at the ROC-determined optimal cut-off level of121 μM/min/mL (HR 2.94 [1.54, 5.62], p=0.001, FIG. 3). After adjustingfor log(BNP), log(hsCRP), Framingham risk factors, and CrCl, lower serumarylesterase levels still maintains a robust 2.6-fold increased risk inthe development of MACE at 3 years (HR 2.59 [1.34, 4.99], p=0.004, Table3; FIG. 3).

TABLE 3 Hazard Ratio 95% confidence ( interval) p value Univariate ModelArylesterase activity 2.94 (1.54, 5.62) 0.001 <121 μM/min/mLMultivariate Model Arylesterase activity 2.59 (1.34, 4.99) 0.004 <121μM/min/mL Log (BNP) 1.25 (1.08, 1.45) 0.003 Log (hsCRP) 1.18 (1.04,1.33) 0.008 Framingham Risk Score 1.05 (0.99, 1.11) 0.09 CreatinineClearance 1.00 (0.99, 1.00) 0.23

Discussion

The major findings of this study is the demonstration of a robustassociation between low PON1 activity measures, as monitored by serumarylesterase activity levels, and poor long-term prognosis in patientswith impaired LV systolic function. The prognostic value of serumarylesterase activity levels within the cohort was both independent of,and stronger than, standard cardiac prognostic indices of diseaseseverity within heart failure patients, such as BNP. There were alsonotable differences in serum arylesterase activity between the studypopulation of stable patients with impaired LV systolic function andthose admitted with advanced decompensated systolic heart failure. Theseobservations are in parallel with reports of prognostic value of otheroxidative stress markers in patients with heart failure, but performedin a large scale using a high-throughput platform. What is unique in thefindings is the potential identification and quantification of animportant anti-oxidative pathway that is broadly affected in the settingof cardiac dysfunction, thereby providing opportunities to modulate orenhance such pathways in treating systolic heart failure.

PON1 activity measurement, such as with arylesterase activity, isreadily detectable in human serum, and low arylesterase activity hasbeen associated with increased systemic measures of oxidative stress,heightened cardiovascular disease risk¹³, and increased disease severityin other end-organ dysfunction^(14,15). Instead of indirectly measuringbyproducts of oxidative stress, quantifying serum arylesterase activitylevels facilitates a direct measurement of the anti-oxidative processthat has been previously described in protecting organophosphatetoxicityl⁶ and reducing atherosclerotic cardiovascular risk inhumans^(13,17) and animal models (Shih D M, Gu L, Xia Y R, Navab M, Li WF, Hama S, Castellani L W, Furlong C E, Costa L G, Fogelman A M, Lusis AJ. Nature. 1998 Jul 16;394(6690):284-7). The ability of arylesteraselevels to predict long-term outcomes independently of standardprognostic markers such as BNP in this population is intriguing,especially when the strength of risk prediction for serum arylesteraseactivity appeared robust. The strong prognostic value of serumarylesterase activity is not linear, as the lowest three quartiles ofsubjects had poorer outcomes compared to the highest quartiles. Thisfinding may imply that a clinical meaningful “cut-off” should beemployed for defining a high-risk cohort, which represented a broadsystolic heart failure population. In contrast, those with relativelypreserved (high) serum arylesterase activity may also be reassured thatthey are in a relatively lower risk category for long-term adversecardiac events.

The precise pathophysiologic mechanism of PON1 activity in human heartfailure has not been previously examined. The expression and variabilityof systemic arylesterase activity over the natural history of heartfailure is largely unknown, although it is conceivable that reducedserum arylesterase activity can adversely contribute to diseaseprogression in a number of ways. The best described mechanism is theassociation between PON1 activity and protection of lipoproteinoxidation that antagonizes progression of atherosclerotic coronaryartery disease, a major cause of heart failure in Western societies.Another potential protective mechanism for arylesterase activity mayinclude the possibility for limiting microvascular dysfunction as aresult of endothelial dysfunction^(18,19). Indeed, lower serumPON1/arylesterase activity has been reported in patients with cardiacsyndrome X²⁰, and genetic polymorphisms favoring preservedPON1/arylesterase are protective of microvascular diseases in patientswith diabetes mellitus²¹.

REFERENCES

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1. A method for assessing a risk of experiencing a major adverse cardiacevent (MACE) in a test subject with heart failure comprising:determining a level of paraoxonase 1 (PON1) activity in a serum ornon-chelated plasma sample from the test subject; and comparing thelevel of PON1 activity in the test subject's serum or non-chelatedplasma sample with a control or baseline value based on levels of PON1activity in serum or non-chelated plasma samples from a population ofcontrol subjects, wherein a test subject whose level of PON1 activity isless than the control or baseline value is at risk of experiencing aMACE.
 2. The method of claim 1, wherein the MACE is selected from thegroup consisting of: non-fatal cerebrovascular accident; non-fatalmyocardial infarction (MI); and death.
 3. The method of claim 2, whereinthe non-fatal cerebrovascular accident is selected from the groupconsisting of: stroke and transient ischemic attacks.
 4. The method ofclaim 1, wherein the test subject's heart failure is selected from thegroup consisting of: systolic heart failure, diastolic heart failure,and left ventricular dysfunction.
 5. The method of claim 1, wherein thePON1 activity is determined by measuring arylesterase, paraoxonase,lipolactonase, or lactonase activity in the subject's serum ornon-chelated plasma sample.
 6. The method of claim 5, wherein thearylesterase, paraoxonase, lipolactonase, or lactonase activity isdetermined by spectrophotometry.
 7. The method of claim 1, wherein thecontrol subjects are apparently healthy control subjects.
 8. The methodof claim 7, wherein the apparently healthy control subjects are withoutany known cardiac diseases.
 9. The method of claim 1, wherein the testsubject whose level of PON1 activity is less than the control orbaseline value is at risk of experiencing a MACE within 3 years.
 10. Themethod of claim 1, wherein the test subject whose level of PON1 activityis less than the control or baseline value is at risk of experiencing aMACE within 10 years.
 11. The method of claim 1, wherein the testsubject whose level of PON1 activity is less than the control orbaseline value is at risk of experiencing a MACE in more than 10 years.12. A method for assessing a risk of developing heart failure in a testsubject comprising: determining a level of PON1 activity in a serum ornon-chelated plasma sample from the test subject; and comparing thelevel of PON1 activity in the test subject's serum or non-chelatedplasma sample with a control or baseline value based on levels of PON1activity in serum or non-chelated plasma samples from a population ofcontrol subjects, wherein a test subject whose level of PON1 activity isless than the control or baseline value is at risk of developing heartfailure.
 13. A method for assessing a risk of requiringrevascularization in a test subject with heart failure comprising:determining a level of paraoxonase 1 (PON1) activity in a serum ornon-chelated plasma sample from the test subject; and comparing thelevel of PON1 activity in the test subject's serum or non-chelatedplasma sample with a control or baseline value based on levels of PON1activity in serum or non-chelated plasma samples from a population ofcontrol subjects, wherein a test subject whose level of PON1 activity isless than the control or baseline value is at risk of requiringrevascularization.
 14. The method of claim 13, wherein the PON1 activityis determined by measuring arylesterase, paraoxonase, lipolactonase, orlactonase activity in the subject's serum or non-chelated plasma sample.15. A method for assessing a risk of requiring a heart transplant in atest subject with heart failure comprising: determining a level ofparaoxonase 1 (PON1) activity in a serum or non-chelated plasma samplefrom the test subject; and comparing the level of PON1 activity in thetest subject's serum or non-chelated plasma sample with a control orbaseline value based on levels of PON1 activity in serum or non-chelatedplasma samples from a population of control subjects, wherein a testsubject whose level of PON1 activity is less than the control orbaseline value is at risk of requiring a heart transplant.
 16. Themethod of claim 15, wherein the PON1 activity is determined by measuringarylesterase, paraoxonase, lipolactonase, or lactonase activity in thesubject's serum or non-chelated plasma sample.
 17. A method forassessing a risk of requiring unscheduled hospitalization for heartfailure in a test subject comprising: determining a level of paraoxonase1 (PON1) activity in a serum or non-chelated plasma sample from the testsubject; and comparing the level of PON1 activity in the test subject'sserum or non-chelated plasma sample with a control or baseline valuebased on levels of PON1 activity in serum or non-chelated plasma samplesfrom a population of control subjects, wherein a test subject whoselevel of PON1 activity is less than the control or baseline value is atrisk of requiring unscheduled hospitalization for heart failure.
 18. Themethod of claim 17, wherein the PON1 activity is determined by measuringarylesterase, paraoxonase, lipolactonase, or lactonase activity in thesubject's serum or non-chelated plasma sample.
 19. A method forassessing a risk of progression of heart failure status in a testsubject comprising: determining a level of paraoxonase 1 (PON1) activityin a serum or non-chelated plasma sample from the test subject; andcomparing the level of PON1 activity in the test subject's serum ornon-chelated plasma sample with a control or baseline value based onlevels of PON1 activity in serum or non-chelated plasma samples from apopulation of control subjects, wherein a test subject whose level ofPON1 activity is less than the control or baseline value is at risk ofprogression of heart failure status.
 20. The method of claim 19, whereinthe PON1 activity is determined by measuring arylesterase, paraoxonase,lipolactonase, or lactonase activity in the subject's serum ornon-chelated plasma sample.
 21. A kit comprising: one or more reagentsfor determining a level of PON1 activity in a subject; and informationfor assessing the subject's risk of at least one of: experiencing aMACE; requiring revascularization; requiring a heart transplant;requiring unscheduled hospitalization for heart failure; and progressionof heart failure status.